WO2010136676A1 - Formulations à compartiments multiples à base de molécules ou macromolécules amphiphiles fonctionnelles - Google Patents
Formulations à compartiments multiples à base de molécules ou macromolécules amphiphiles fonctionnelles Download PDFInfo
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- 0 *C[C@@]1OC([n]2c3ncnc(N)c3nc2)=CC1O Chemical compound *C[C@@]1OC([n]2c3ncnc(N)c3nc2)=CC1O 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the invention relates to novel multi-compartmental formulations based on functional amphiphilic molecules or macromolecules for the transport or vectorization of at least one therapeutic agent, in particular an anti-tumor agent, as well as their method of preparation, and their uses.
- a therapeutic agent in particular an anti-tumor agent
- cis-platinum is a widely used anti-tumor agent, in particular for the treatment of solid tumors.
- its use is limited by its toxicity and the appearance of acquired resistance.
- US Pat. No. 6,001,817 describes compositions containing cisplatin and a vector comprising at least one nucleoside or deoxynucleoside.
- US Pat. No. 7,908,160 relates to ligand-bound cis-platinum derivatives whose activity is reversible as a function of ligand binding.
- WO01 / 32139 describes cis-platinum compositions encapsulated in lipid nanoparticles obtained by repeated cycles of heating and freezing, based on negatively charged natural lipids, in particular dioleylphosphatidylserine. It is stated in this application that cis-platinum forms positively charged aggregates in water with a higher solubility than uncharged species, allowing their interaction with negatively charged lipid membranes and reorganization of membranes. lipids around cis-platinum aggregates.
- therapeutic agents in particular anti-tumor agents.
- each of the compartments can serve as a reservoir.
- multi-compartmental formulations formed from functional amphiphilic molecules or macromolecules, have improved stability properties, particularly at 37 ° C, allowing prolonged vectorization over time of said therapeutic agents and allow intracellular delivery. effective and fast therapeutic agents.
- the invention therefore has, according to a first aspect, a nanoparticle-based multi-compartment formulation consisting of a solid core containing a therapeutic agent, surrounded by at least two lipid layers of different polarity formed from molecules or macromolecules. functional amphiphiles.
- nanoparticle is meant a particle having an average diameter of about 1 to 200 nm, preferably 25 to 150 nm.
- nanoparticle according to the invention or “multi-compartment nanoparticle” is understood to mean a multi-compartment formulation in the form of a nanoparticle consisting of a solid core containing a therapeutic agent, surrounded by at least two Lipid layers of different polarity formed from functional amphiphilic molecules or macromolecules.
- each lipid layer constitutes a compartment which may comprise a therapeutic agent identical to or different from that present in the heart.
- the multi-compartment formulations according to the invention are formed from said lipid layers of different polarity in the presence of the therapeutic agent (s) and not from a pre-formed particle, which allows a "à la carte” encapsulation of the active ingredient in the desired compartment (s), depending on the desired activity.
- This particular structure gives the nanoparticles with multiple compartments according to the invention a stability (shelf life) compatible with the delivery of a therapeutic agent, and allows their disintegration after release of this therapeutic agent.
- the first lipid layer will consist of one or more anionic lipid (s) and the second lipid layer will consist of one or more cationic lipid (s).
- each successive lipid layer surrounding the core consists of lipids different from the previous one and that each layer has a global surface charge either negative (consisting of anionic lipids) or positive (consisting of cationic lipids ), or neutral (consisting of neutral lipids).
- a first lipid layer may be formed of anionic lipids and will carry a negative surface charge
- the second lipid layer may be formed of cationic lipids and will carry a positive surface charge.
- each of the layers is measurable by their zeta potential, for example, according to the technique described in Andréa Mayer et al. Toxicology, 2009, 258, 139-147 or K. Furusawa and K. Uchiyama, 1988, 140, 217-226.
- the nanoparticles with multiple compartments according to the invention can comprise an alternation of layers anionic and cationic lipids and an outer layer consisting of one or more neutral lipids.
- each lipid layer consists of at least one functional amphiphilic compound of formula (I)
- X represents an oxygen atom, sulfur atom or a methylene group
- B represents a purine or pyrimidine base such as uracil, adenine, guanine, cytosine, thymine, hypoxanthine, or their derivatives, or a non-natural mono- or bi-cyclic heterocyclic base of which each ring comprises 4 to 7 ring members, optionally substituted;
- L 1 and L 2 which may be identical or different, represent hydrogen, an oxycarbonyl group -O-C (O) -, a thiocarbamate group -OC (S) -NH-, a carbonate group -O-C (O) - O-, a carbamate group -O-C (O) -NH-, an oxygen atom, a phosphate group, a phosphonate group or a heteroaryl group comprising 1 to 4 nitrogen atoms, substituted or unsubstituted by a chain hydrocarbonaceous, saturated or unsaturated, linear or branched C 2 -
- L 1 or L 2 represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, unsubstituted or substituted by a linear or branched C 2 -C 30 alkyl chain ; - R 1 and R 2 , identical or different, represent
- a linear or branched C 2 -C 30 hydrocarbon-based chain preferably in C 6 -C 25, especially C 8 -C 2 S, saturated or partially unsaturated, optionally totally or partially fluorinated, unsubstituted or substituted on the chain end carbon with a fluorine atom or with a benzyl ester or ether or naphthyl, or - a diacyl chain in which each C 2 -C 30 acyl chain, or
- L 1 or L 2 represents hydrogen and the other represents a hydroxyl group or a heteroaryl group comprising 1 to 4 nitrogen atoms, R 1 and R 2 do not exist;
- R 3 represents
- R 4 and R 6 identical or different, represent an atom of hydrogen or a linear or branched alkyl chain of linear or branched C 1 -C 5 or hydroxyalkyl
- V is -O-, -S-, or -NH-
- R 7 is H or
- R 3 is covalently linked to another substituent R 3 , identical or different, from another compound of formula (I), identical or different, to form a compound of formula (I) in the form of dimer, and each lipid layer has a polarity different from that of the previous one.
- the charge of the compounds of formula (I) is determined by the polar groups they contain, these being essentially present in or consisting of the substituents L 1 , L 2 and / or R 3 .
- Anionic compounds of formula (I) that can be used to prepare the first lipid layer can be, for example, chosen from anionic nucleolipids such as compounds of formula (I) in which Li, L 2 and / or R 3 represent a charged group. negatively such as, for example, an optionally substituted phosphate, phosphonate, carboxylate, sulfate, etc. group.
- Compounds of formula (I) cationic usable for preparing the first lipid layer may be, for example, selected from cationic nucleolipids such as compounds of formula (I) in which Li, L 2 and / or R 3 represent a charged group positively such as, for example, an ammonium group, phosphonium, imidazolium, etc., optionally substituted.
- the charge of these polar groups may also vary depending on the pKa of these groups, for example when it is an amino group, imidazole, phosphate, etc.
- therapeutic agent is meant, for example, a natural or synthetic molecule used for the prevention or treatment of a pathology or the restoration of a biological function, in vitro or in vivo. in particular in animals, including humans, or isolated cells, with the exception of nucleic acids or fragments thereof.
- a molecule may be chosen, for example, from the active ingredients of medicaments, in particular from anti-tumor agents such as, for example:
- platinum complexes among which may especially be mentioned cis-platinum, carboplatin, oxaliplatin, nedaplatin, lobaplatin, etc., or
- iron derivatives such as, for example, ferrocenium salts, iron-containing nucleoside analogues, iron (II) complexes containing pyridyl pentadenate ligands, or
- cobalt derivatives such as, for example, hexacarbonyl-dicobalt complexes, alkyne-cobalt complexes, the Co (III) complex containing a bicyclic nitrogen ligand, or
- gold derivatives such as, for example, Auranofin, gold complexes (I), (III) and (III), aurothioglucose, etc.
- the multi-compartment formulation according to the invention makes it possible to encapsulate these molecules and to ensure their intracellular delivery while limiting the phenomena of resistance acquired to these compounds.
- Platinum complexes, in particular cis-platinum, are preferred therapeutic agents for the purpose of the invention.
- Inorganic complexes based on ruthenium II and / or III may be, for example, the complexes called NAMI-A, RAPTA-C, KP1019.
- Such non-platinum complexes are described in Ott I. and Gust R., Arch. Pharm. Chem. Life Sci. 2007, 340, 117-126; Reedijk J., Curr Opin Chem Biol., 1999, 3, 236-40; Haimei Chen et al., J. Am. Chem. Soc., 2003, 125, 173-186.
- the nanoparticles thus obtained exhibit a structure allowing an efficient and rapid intracellular delivery of encapsulated active ingredients, in particular anti-tumor agents.
- the intermolecular interactions of the compounds of formula (I) induce an increase in the cohesion forces on the surface of the nanoparticles, which results in greater stability in time, under the conditions of use.
- the multi-compartment structure of the nanoparticles according to the invention based on multiple lipid layers having a flexible polarity, gives them numerous advantages, in particular:
- nanoparticles also have a shelf life compatible with their use as a therapeutic agent vector.
- n is advantageously between
- 1 and 500 preferably between 1 and 100, in particular between 1 and 50, most particularly between 1 and 10.
- linear or branched C 1 -C 5 alkyl is meant for example a methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl or tert-butyl radical, preferably methyl or ethyl.
- the purine or pyrimidine base, or the non-natural heterocyclic base may be substituted with at least one substituent selected from, for example, a halogen, an amino group, a carboxy group, a carbonyl group, carbonylamino group, hydroxy, azido, cyano, alkyl, cycloalkyl, perfluoroalkyl, alkyloxy (for example, methoxy), oxycarbonyl, vinyl, ethynyl, propynyl, acyl and so on.
- substituent selected from, for example, a halogen, an amino group, a carboxy group, a carbonyl group, carbonylamino group, hydroxy, azido, cyano, alkyl, cycloalkyl, perfluoroalkyl, alkyloxy (for example, methoxy), oxycarbonyl, vinyl, ethynyl, propynyl, acyl and so on.
- non-natural heterocyclic base means a base other than uracil, adenine, guanine, cytosine, thymine or hypoxanthine, which does not exist in nature.
- heteroaryl group containing 1 to 4 nitrogen atoms is meant a monocyclic or bycyclic carbocyclic group, aromatic or partially unsaturated, containing 5 to 12 atoms, interrupted by 1 to 4 nitrogen atoms, in particular the pyrazole groups, triazole, tetrazole or imidazole.
- the invention also relates, in a further aspect, to a process for preparing a solid nanoparticle solid compartment-containing multi-compartment formulation comprising a therapeutic agent-containing heart surrounded by at least two lipid layers of different polarity formed from functional amphiphilic molecules or macromolecules, in which each lipid layer constitutes a compartment likely to comprise a therapeutic agent identical to or different from that present in the heart, comprising the following steps: a) preparing a mixture of at least one functional amphiphilic molecule or macromolecule, in particular an amphiphilic compound functional group of formula (I) as defined above, and a therapeutic agent, b) subjecting said mixture to repeated cycles of heating and freezing, so as to obtain nanoparticles containing said therapeutic agent, and c) recover the nanoparticles containing said therapeutic agent thus obtained, d) bringing said nanoparticle into presence with at least one functional amphiphilic molecule or macromolecule, in particular a functional amphiphilic compound of formula (I) as defined above, having a polarity different from
- the therapeutic agent is an anti-tumor agent, in particular a platinum complex, in particular cis-platinum.
- the steps of the method may be repeated the number of times necessary to obtain the desired number of lipid layers.
- an additional step consisting in the formation of a neutral lipid layer consisting of at least one functional amphiphilic molecule or macromolecule, in particular a functional amphiphilic compound of formula (I) as defined above said molecule or said compound of formula (I) being neutral, may be carried out between step d) and step e).
- colipid in addition to the functional amphiphilic compound, at least one colipid will be used.
- colipid is meant a compound used in combination with the compound of formula (I), which participates in the development of the structure of the lipid layer (s) of the nanoparticle.
- a zwitterionic colipid will preferably be used.
- Said colipid may be, for example, chosen from dioleylphosphatidylcholine (DOPC), dioleylphosphatidyluridinephosphatidylcholine (DOUPC) or dioleylphosphatidylethanolamine (DOPE).
- DOPC dioleylphosphatidylcholine
- DOUPC dioleylphosphatidyluridinephosphatidylcholine
- DOPE dioleylphosphatidylethanolamine
- These compounds can act as colipids when used in admixture with a compound of formula (I).
- they can be included in formula (I), such as, for example, dioleylphosphatidyluridinephosphatidylcholine (SOHC).
- SOHC dioleylphosphatidyluridinephosphatidylcholine
- they will play either the role of compound of formula (I) or, in combination with another compound of formula (I), the role of colipid.
- step d) a therapeutic agent identical to or different from that used in step a).
- amphiphilic compound (s) functional (s) of formula (I) used in step a) is (are) anionic (s) and the amphiphilic compound (s) (s) functional (s) of formula (I) used in step d) is (are) cationic (s).
- a functional amphiphilic compound of formula (I) to form the outermost lipid layer, which may be carried out between step d) and step e).
- the process for the preparation of the multi-compartment formulations may comprise the steps implemented under the following general conditions, which illustrate, for example, the obtaining of a nanoparticulate multi-compartment formulation consisting of a core solid containing a therapeutic agent, surrounded by two lipid layers of different polarity formed from compounds of formula (I):
- the desired quantity of therapeutic agent preferably an anti-tumor agent, is dissolved in distilled water;
- the first lipid film is rehydrated in the therapeutic agent solution, preferably an anti-tumor agent.
- the therapeutic agent solution preferably an anti-tumor agent.
- a clear solution is obtained by sonification and heating;
- the solution is rapidly cooled, for example by immersion in liquid nitrogen.
- This heating / cooling cycle is preferably carried out from 1 to 10 times, in particular from 5 to 10 times, in particular 10 times;
- nanoparticles comprising a core rich in therapeutic agent and two lipid layers of different polarity
- a second lipid film is prepared from a compound of formula (I) of a different polarity from that of the compound of formula (I) used in the first part of the process;
- the second lipid film is rehydrated with the previously recovered supernatant
- the pellet after centrifugation, the pellet is discarded and the supernatant is recovered, containing the nanoparticles with multiple compartments comprising two lipid layers of different polarity.
- the steps of the above method are repeated for the number of times necessary to obtain the desired number of lipid layers.
- the number of lipid layers will be between 2 and 6.
- an additional step consisting of the formation of a lipid layer consisting of at least one functional amphiphilic compound of formula (I) as defined above, said compound of formula (I) being neutral, may be carried out during the 2nd part of the process, before the final step for the recovery of a multiple compartment nanoparticles according to the invention.
- At least one colipid, as defined will be used. upper.
- Preferred formulations according to the invention are those in which the first lipid layer is anionic and the second lipid layer is cationic.
- the organic solvent may be chosen, for example, from organic solvents customary in the field, such as, for example, chloroform or dichloromethane, an alcohol such as methanol or ethanol, etc.
- the heating is preferably carried out at a temperature of the order of 20 ° C. to 80 ° C., and the cooling is carried out at a temperature of about -190 ° C. (liquid nitrogen) at 0 ° C. (ice) .
- a suitable heating / cooling cycle may, for example, be 45 ° C for heating and -78 ° C for cooling.
- the therapeutic agent is chosen from platinum complexes (cis-platinum, carboplatin, etc.), cis-platinum being particularly preferred, or ruthenium capable of binding to platinum complexes, or else platinum-free inorganic complexes based on ruthenium II or III, titanium, gallium, cobalt, iron or gold mentioned above.
- platinum complexes cis-platinum, carboplatin, etc.
- cis-platinum being particularly preferred
- ruthenium capable of binding to platinum complexes
- platinum-free inorganic complexes based on ruthenium II or III, titanium, gallium, cobalt, iron or gold mentioned above.
- the nanoparticles obtained may optionally be extruded on a polycarbonate filter having, for example, a pore diameter of the order of 100 or 200 nm.
- Multi-compartment nanoparticles consist of a solid core rich in therapeutic agent (active principle) surrounded by at least two lipid layers of different polarity constituted by functional amphiphilic compound of formula (I) as defined above, with or without co-lipid.
- said lipid mixture contains only at least one compound of formula (I) and does not contain colipid.
- the therapeutic agent will preferably be used at a concentration of the order of 0.1 ng / ml to 10 mg / ml in the aqueous phase, so that the intracellular delivery of the active principle is important.
- Preferred compounds of formula (I) useful for forming a lipid layer are those wherein X is oxygen.
- L 1 , L 2 and / or R 3 represent a positively charged group such as, for example, an optionally substituted ammonium, phosphonium, imidazolium, etc. group, are preferred compounds for obtain a cationic lipid layer.
- the invention relates to the nanoparticles with multiple compartments as defined above comprising these compounds of formula (I) and a therapeutic agent, in particular an antitumor agent, in particular platinum complexes (such as, for example cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin,), or ruthenium capable of binding platinum complexes, or inorganic complexes without platinum ruthenium, titanium, gallium, cobalt, iron or gold mentioned above.
- platinum complexes such as, for example cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin,
- ruthenium capable of binding platinum complexes, or inorganic complexes without platinum ruthenium, titanium, gallium, cobalt, iron or gold mentioned above.
- Cisplatin is a preferred anti-tumor agent for the purpose of the invention.
- the compounds of formula (I) may also contain purine or pyrimidine base derivatives having antineoplastic activity, such as, for example, aracytosine (AraC), 5-fluorouracil (5-FU), Iododeoxyuridine (IdU ), 2'-deoxy-2'-methylidenecytidine (DMDC) or 5-chloro-6-azido-5,6-dihydro-2'-deoxyuridine.
- the subject of the invention is also the use of the multi-compartment nanoparticles described above, as an agent for the transport or vectorization of therapeutic agents, in particular antitumor agents.
- the invention relates to the use of the multi-compartment nanoparticles described above as an agent for the intracellular delivery of therapeutic agents, particularly antitumor agents.
- the invention also relates to the use of the multi-compartment nanoparticles described above, for the preparation of anti-tumor drugs.
- the invention also relates to the nanoparticles with multiple compartments described above, for the treatment of tumoral diseases, in particular of cancers, such as, for example, ovarian, testicular, colon, cervix and ovarian cancers. uterus, lung, or adenosarcoma etc.
- cancers such as, for example, ovarian, testicular, colon, cervix and ovarian cancers. uterus, lung, or adenosarcoma etc.
- Said nanoparticles with multiple compartments can be obtained by the method described above.
- the invention also relates to pharmaceutical compositions comprising nanoparticulate multi-compartment formulations consisting of a solid core containing a therapeutic agent, surrounded by at least two lipid layers of polarity. different formed from functional amphiphilic molecules or macromolecules (or nanoparticles with multiple compartments), as described above, and a pharmaceutically acceptable vehicle.
- the mixture is then oxidized by adding 43 ml of a solution of 0.02M diiod in THF / Pyr / H 2 O. After 12 h at room temperature, the solvent is evaporated in vacuo. The residue is dissolved in 8 mL of dichloromethane. Then 0.2 mL of 1, 5- diazabicyclo [5.4.0] undec-7-ene (DBU) (1.3 eq, 0.87 mmol) are added to the reaction medium for 5 h. The reaction medium is washed with a 0.1N solution of HCl and then with a saturated solution of Na 2 S 2 O 7 . The organic phase is concentrated under vacuum. The compound is obtained after purification by flash chromatography (381 mg) using an elution gradient (MeOH / DCM 9: 1 to 1: 1).
- the reaction medium is stirred magnetically for 24 hours at room temperature and under nitrogen.
- the mixture is then oxidized by adding 43 ml of a 0.02M solution of diiodin in THF / Pyr / H 2 O. After 12 hours at room temperature, the solvent is evaporated under vacuum and dried at the pump under P 2 O 5. for one night. The residue is dissolved in 8 mL of dichloromethane. Then, 0.2 ml of 1,5-diazabicyclo [5.4.0] undec-7-ene (DBU) (1.3 eq, 0.87 mmol) are added to the reaction medium for 5 h. The reaction medium is washed with a 0.1N solution of HCl and then with a saturated solution of Na 2 S 2 Oa.
- DBU 1,5-diazabicyclo [5.4.0] undec-7-ene
- Solution A 20 mg of diC16dT are solubilized in 2 mL of dichloromethane (10 mg / mL). This sample is stored at -20 ° C.
- Solution B DOPC: 20 mg / mL solution in dichloromethane stored at -20 ° C.
- Solution C DOTAU: solution at 20 mg / mL in dichloromethane stored at -20 ° C.
- the suspension is stirred and placed in a glass hemolysis tube and sonicated for 7 minutes. After sonication, the suspension is centrifuged at 10,000 rpm / 5min / 20 ° C. The supernatant is discarded and the pellet of the nanoparticles is resuspended in 1 ml of milliQ water. This step is renewed a second time.
- the suspension is centrifuged at 1000 rpm / 2.5 min / 20 ° C.
- the pellet is discarded and the supernatant contains the anionic nanoparticles.
- the zeta potential measured according to the technique described in Andréa
- Vortex stirring for 5 minutes is performed followed by sonication for one minute.
- the suspension is centrifuged at 10,000 rpm for 5 min at 20 ° C to remove lipids unrelated to the nanoparticles. the supernatant is removed and the pellet is rehydrated with 1 mL of milliQ water.
- the suspension is centrifuged at 1000 rpm / 2.5 min / 20 ° C. The pellet is discarded and the supernatant contains the multi-compartment nanoparticles with cationic surface layer.
- the zeta potential measured as before, is
- Example 11 Stability test (% Cis-platinum released)
- the nanoparticles prepared according to the protocol of Example 10 are assayed in optical ICP (the measured value corresponds to the total concentration).
- the suspension of the nanoparticles is aliquoted in 5 tubes eppendorf ® (150 ⁇ L). The latter are incubated at 37 ° C. with shaking (300 rpm) for different times (0, 2.5, 5, 10 and 24 h).
- the tube is centrifuged at 14000 rpm / 10min / 20 ° C and 50 ⁇ l of supernatant (recovered gently so as not to resuspend the pellet) are assayed.
- DOPS 1,2-dioleoyl-sn-glyero-3-phosphocholine
- DOPC 1,2-dioleoyl-sn-glyero-3-phosphocholine
- Ct total concentration found without incubation and without centrifugation.
- nanoparticles comprising a single anionic lipid layer obtained at the end of step 4 of Example 10 (denoted NP-) are represented by the symbol - * -, the nanoparticles according to the invention (noted
- NP + comprising a first anionic lipid layer and a second cationic lipid layer obtained at the end of step 6 of Example 10 are represented by the symbol -m- and the nanoparticles based on DOPC / DOPS (denoted PS) by the symbol - ⁇ * - -
- the nanoparticles prepared according to the protocol of Example 10 are assayed in optical ICP (the measured value corresponds to the total concentration).
- the suspension of the nanoparticles is aliquoted in 5 eppendorf® tubes (150 ⁇ L). The latter are centrifuged at 10,000 rpm for 5 min at 20 ° C. 50 ⁇ l of the supernatant is assayed by optical ICP and the remaining 100 ⁇ l are discarded.
- the pellet containing the nanoparticles is rehydrated with 150 ⁇ l of fetal calf serum (FCS, ref invitrogeni 0270-106). The samples are incubated at 37 ° C. with shaking (300 rpm) for different times (0, 2.5, 5, 10 and 24 h). At a given time (x), the tube is centrifuged at a given time (x), the tube is centrifuged at
- DOPS 1,2-Dioleoyl-sn-glycero-3- [phospho- L-serine] based nanoparticles with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as colipid are prepared according to the same protocol for comparison.
- Ct total concentration found without incubation and without centrifugation.
- nanoparticles according to the invention comprising a first anionic lipid layer and a second lipid layer Cationic obtained in Example 10 are represented by the symbol -B- and the nanoparticles based on DOPC / DOPS (denoted PS) by the symbol - ⁇ -
- Example 13 Intracellular cisplatin dosage
- Protocol IGROV1 cells (ovarian adenocarcinoma line sensitive to cis platinum) at 80% confluency (10 cm diameter box) are treated with 100 ⁇ M of free cisplatin or encapsulated in the nanoparticles of Example 10 for 2 , 4 or 6 h. At the end of this treatment, two washes with PBS are carried out. The cells are treated with trypsin and resuspended in PBS. Two PBS washes of cell suspensions were performed (centrifugation 1000 rpm / 1 min). The cells are suspended in 1 mL of PBS and counted.
- 10 6 cells are lysed with 500 ⁇ l of the cell lysis solution (lysis buffer from SIGMA). The volume is supplemented with milliQ water at 1% HNO 3 acid to reach 5 mL.
- FIG. 3 shows the concentration of cis-platinum released after cell lysis (expressed in nanomole / 10 6 cells / 100 ⁇ M of treatment) as a function of time, corresponding to the concentration of internalized cisplatin in the treated cells.
- the study consists in determining the concentration inhibiting 50% of cell proliferation (Cl 50 ) on a panel of tumor lines, namely
- A2780 Human ovarian carcinoma epithelial tumor sensitive to cis-platinum and A2780 / CisPt resistant to cisplatin (Human ovarian carcinoma epithelial tumor)
- L1210 (mouse lymphocytic leukemia) sensitive to cis-platinum
- L1210 / CisPt (mouse lymphocytic leukemia) resistant to cisplatin (human leukemia)
- OVCAR3 ovarian carcinoma
- - P388 human lymphoma
- 2500 cells ovarian adenocarcinoma lines, etc.
- the medium is aspirated and the cells are treated with cisplatin free or encapsulated in the nanoparticles of Example 10 in 100 ⁇ L of medium without serum at different concentrations (500, 100, 10, 1, 0.1, 0.01 0.001 ⁇ M).
- the medium is removed and the cells are washed twice with 100 ⁇ l of PBS and then incubated with 100 ⁇ l of the medium with serum.
- cell viability is revealed by adding 20 ⁇ L of MTS.
- the absorbance at 490 nm is measured after 2 to 4 hours of incubation at 37 ° C. Absorbance is proportional to cell viability.
- FIG. 4 shows the concentration necessary to obtain 50% of cell death (IC 50) with free cis-platinum (light gray column on the left) or the nanoparticles according to the invention comprising a first anionic lipid layer. and a second cationic lipid layer obtained at the end of step 6 of Example 10 (denoted by NP +) containing cis-platinum (dark gray column on the right).
- the nanoparticles according to the invention are respectively 18 and 140 times more effective than free cisplatin on the A2780 sensitive and A2780 resistant lines.
- 2500 cells (IGROV1, SKOV3, ovarian adenocarcinoma lines) per well are incubated in 100 ⁇ l of the medium with serum. After 24 h the medium is aspirated and the cells are treated with cisplatin free or encapsulated in the nanoparticles of Example 10 in 100 ⁇ l of serum-free medium at different concentrations (500, 100, 10, 1, 0.1, 0.01 0.001 ⁇ M). After 24 hours of treatment, the medium is removed and the cells are washed twice with 100 ⁇ l of PBS and then incubated with 100 ⁇ l of medium with serum.
- cell viability is revealed by adding 20 ⁇ L of MTS.
- the absorbance at 490 nm is measured after 2 to 4 hours of incubation at 37 C C.
- the absorbance is proportional to cell viability.
- FIG. 5 shows the concentration necessary to obtain 50% of cell death (IC50) with free cis-platinum (column 1, left), the DOPC / DOPS-based control nanoparticles ( noted PS) (column 2, center left), the nanoparticles comprising a single anionic lipid layer obtained at the end of step 4 of Example 10 (denoted NP-) (column 3, center right) and the nanoparticles according to the invention (noted NP +) comprising a first anionic lipid layer and a second cationic lipid layer obtained at the end of step 6 of Example 10 (right column).
- Figure 5A relates to the IGROV1 cell line
- Figure 5B relates to the SKOV3 cell line.
- NP + nanoparticles containing cisplatin according to the invention are more effective than free cisplatin in the two cell lines, IGROV1 (sensitive to cisplatin) and SKOV3 (cisplatin-resistant).
- nanoparticles containing cisplatin according to the invention are respectively 13 and 14 times more effective than free cisplatin on the lines IGROV1 and SKOV3 respectively.
- Nanoparticle formulations were prepared with different markers, in order to study, on the one hand, the location of the marker in the lipid layer, and, on the other hand, the state of the nanoparticles after their entry into the cells.
- Lipophilic fluorescent probes have been inserted into the formulations as a marker, on the one hand, and as a lipid compound mimicking a prodrug (e.g., a lipid conjugate analogous to an anticancer nucleoside such as 5-FU). ) on the other hand.
- a prodrug e.g., a lipid conjugate analogous to an anticancer nucleoside such as 5-FU.
- Formulation A diC16dT / DOPC 50/50
- Formulation B DOTAU / DOPC 50/50
- the NP1, NP2 and NP3 nanoparticles according to the invention were thus prepared of different compositions, defined as follows:
- the nanoparticles NP1, NP2 and NP3 are represented in FIG. 6.
- the white layer represents an unlabeled lipid layer
- the gray layer represents the fluorescein-labeled layer (formulation C)
- the black layer represents the layer. labeled with rhodamine (formulation D)
- the dashed center represents incorporated cis-platinum.
- the FACS data obtained show the presence of the two fluorescent markers (fluorescein, rhodamine) in the SKV03 cells after incubation in the presence of the NP3 nanoparticles bearing these markers. These results show, on the one hand, that the nanoparticles according to the invention have a multi-compartment structure, and, on the other hand, remain intact after internalization in the cells. Fluorescence microscopy experiments confirmed the results obtained by FACS. The images show that NP1, NP2 and NP3 labeled nanoparticles are internalized intact in SKVO3 cells.
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Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2763139A CA2763139A1 (fr) | 2009-05-29 | 2010-05-28 | Formulations a compartiments multiples a base de molecules ou macromolecules amphiphiles fonctionnelles |
EP10727016.7A EP2435031B1 (fr) | 2009-05-29 | 2010-05-28 | Formulations à compartiments multiples à base de molécules ou macromolécules amphiphiles fonctionnelles |
US13/322,986 US9050268B2 (en) | 2009-05-29 | 2010-05-28 | Functional amphipilic molecule or macromolecule formulations with multiple compartments |
JP2012512415A JP5913094B2 (ja) | 2009-05-29 | 2010-05-28 | 複数区画を有する機能性両親媒性分子又は巨大分子製剤 |
RU2011153706/15A RU2011153706A (ru) | 2009-05-29 | 2010-05-28 | Многосекционные композиции на основе амфифильных функциональных молекул или макромолекул |
BRPI1016064A BRPI1016064A2 (pt) | 2009-05-29 | 2010-05-28 | "formulação com compartimentos múltiplos, processo para a preparação da mesma, agentes de transporte ou de vetorização, e de liberação intracelular de agentes terapêuticos, utilização de uma formulação, e composição farmacêutica" |
ES10727016.7T ES2556635T3 (es) | 2009-05-29 | 2010-05-28 | Formulaciones de compartimentos múltiples a base de moléculas o macromoléculas anfifílicas funcionales |
CN201080031390.9A CN102458379B (zh) | 2009-05-29 | 2010-05-28 | 基于官能两亲分子或大分子的具有多隔室的配制剂 |
IL216623A IL216623A (en) | 2009-05-29 | 2011-11-27 | Functional amphiphilic molecule or macromolecular assemblies with multiple cells |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0902607 | 2009-05-29 | ||
FR0902607A FR2945946B1 (fr) | 2009-05-29 | 2009-05-29 | Formulations a compartiments multiples a base de molecules ou macromolecules amphiphiles fonctionnelles |
Publications (1)
Publication Number | Publication Date |
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WO2010136676A1 true WO2010136676A1 (fr) | 2010-12-02 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/FR2010/000401 WO2010136676A1 (fr) | 2009-05-29 | 2010-05-28 | Formulations à compartiments multiples à base de molécules ou macromolécules amphiphiles fonctionnelles |
Country Status (11)
Country | Link |
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US (1) | US9050268B2 (fr) |
EP (1) | EP2435031B1 (fr) |
JP (1) | JP5913094B2 (fr) |
CN (1) | CN102458379B (fr) |
BR (1) | BRPI1016064A2 (fr) |
CA (1) | CA2763139A1 (fr) |
ES (1) | ES2556635T3 (fr) |
FR (1) | FR2945946B1 (fr) |
IL (1) | IL216623A (fr) |
RU (1) | RU2011153706A (fr) |
WO (1) | WO2010136676A1 (fr) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3085360A1 (fr) | 2015-04-20 | 2016-10-26 | Universite De Bordeaux | Compositions de nanovecteurs à base de lipide chargées avec des nanoparticules métalliques et agent thérapeutique |
EP3502684A1 (fr) | 2017-12-22 | 2019-06-26 | Universite De Bordeaux | Procédé de détection d'ions métalliques dans des solutions aqueuses au moyen de composés nucléolipides |
WO2019162633A1 (fr) | 2018-02-22 | 2019-08-29 | Universite de Bordeaux | Procédé de décontamination d'un milieu liquide aqueux contenant des micropolluants ou d'une surface contaminée par des micropolluants |
WO2023135299A1 (fr) | 2022-01-17 | 2023-07-20 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Nanoparticules solides oligonucléotidiques de nucléolipides pour lutter contre la résistance aux antibiotiques |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2924430B1 (fr) * | 2007-11-30 | 2010-03-19 | Univ Bordeaux 2 | Procede de preparation de nanoparticules a base de molecules ou macromolecules amphiphiles fonctionnelles et leur utilisation |
MD4307C1 (ro) * | 2013-11-28 | 2015-05-31 | Технический университет Молдовы | Metodă de stimulare a motilităţii tractului gastro-intestinal |
US11981703B2 (en) | 2016-08-17 | 2024-05-14 | Sirius Therapeutics, Inc. | Polynucleotide constructs |
US11597744B2 (en) | 2017-06-30 | 2023-03-07 | Sirius Therapeutics, Inc. | Chiral phosphoramidite auxiliaries and methods of their use |
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- 2009-05-29 FR FR0902607A patent/FR2945946B1/fr not_active Expired - Fee Related
-
2010
- 2010-05-28 BR BRPI1016064A patent/BRPI1016064A2/pt not_active IP Right Cessation
- 2010-05-28 JP JP2012512415A patent/JP5913094B2/ja not_active Expired - Fee Related
- 2010-05-28 EP EP10727016.7A patent/EP2435031B1/fr active Active
- 2010-05-28 CN CN201080031390.9A patent/CN102458379B/zh not_active Expired - Fee Related
- 2010-05-28 RU RU2011153706/15A patent/RU2011153706A/ru not_active Application Discontinuation
- 2010-05-28 WO PCT/FR2010/000401 patent/WO2010136676A1/fr active Application Filing
- 2010-05-28 ES ES10727016.7T patent/ES2556635T3/es active Active
- 2010-05-28 CA CA2763139A patent/CA2763139A1/fr not_active Abandoned
- 2010-05-28 US US13/322,986 patent/US9050268B2/en not_active Expired - Fee Related
-
2011
- 2011-11-27 IL IL216623A patent/IL216623A/en not_active IP Right Cessation
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3085360A1 (fr) | 2015-04-20 | 2016-10-26 | Universite De Bordeaux | Compositions de nanovecteurs à base de lipide chargées avec des nanoparticules métalliques et agent thérapeutique |
WO2016170010A1 (fr) | 2015-04-20 | 2016-10-27 | Universite de Bordeaux | Compositions de nanovecteurs à base de lipides chargées de nanoparticules métalliques et d'un agent thérapeutique |
EP3502684A1 (fr) | 2017-12-22 | 2019-06-26 | Universite De Bordeaux | Procédé de détection d'ions métalliques dans des solutions aqueuses au moyen de composés nucléolipides |
WO2019122420A1 (fr) | 2017-12-22 | 2019-06-27 | Universite de Bordeaux | Procédé de détection d'ions métalliques dans des solutions aqueuses à l'aide de composés nucléolipidiques |
WO2019162633A1 (fr) | 2018-02-22 | 2019-08-29 | Universite de Bordeaux | Procédé de décontamination d'un milieu liquide aqueux contenant des micropolluants ou d'une surface contaminée par des micropolluants |
WO2023135299A1 (fr) | 2022-01-17 | 2023-07-20 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Nanoparticules solides oligonucléotidiques de nucléolipides pour lutter contre la résistance aux antibiotiques |
Also Published As
Publication number | Publication date |
---|---|
ES2556635T3 (es) | 2016-01-19 |
BRPI1016064A2 (pt) | 2019-09-24 |
US9050268B2 (en) | 2015-06-09 |
FR2945946A1 (fr) | 2010-12-03 |
FR2945946B1 (fr) | 2011-08-26 |
IL216623A0 (en) | 2012-02-29 |
US20120070505A1 (en) | 2012-03-22 |
RU2011153706A (ru) | 2013-07-10 |
EP2435031A1 (fr) | 2012-04-04 |
JP5913094B2 (ja) | 2016-04-27 |
CN102458379B (zh) | 2014-05-14 |
IL216623A (en) | 2015-03-31 |
CN102458379A (zh) | 2012-05-16 |
EP2435031B1 (fr) | 2015-09-16 |
CA2763139A1 (fr) | 2010-12-02 |
JP2012528133A (ja) | 2012-11-12 |
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